Phenolic compounds, antioxidant activity and sensory evaluation of sea buckthorn (Hippophae rhamnoides L.) leaf tea

Abstract Sea buckthorn leaf tea, an emerging potential functional beverage product, has not yet had appropriate product standards and corresponding quality evaluation methods, and its poor taste directly affects the acceptance of the population, thus limiting its market consumption potential. In this study, two major packaging forms of sea buckthorn leaf tea available in the Chinese market were selected. The contents of total phenolics, total flavonoids, and 10 phenolic compounds, as well as the in vitro antioxidant capacity and sensory characteristics of sea buckthorn leaf tea were analyzed. Results showed that the quality of sea buckthorn leaf tea in the Chinese market varied widely. The total phenolic content, total flavonoid content, antioxidant activity, and consumer acceptance of bagged sea buckthorn leaf tea were higher than those of bulk sea buckthorn leaf tea. Multifactorial statistical analysis showed that the taste astringency of sea buckthorn leaf tea was closely related to ellagic acid and isorhamnetin‐3‐O‐neohesperidin. Furthermore, isorhamnetin‐3‐O‐neohesperidin had a greater effect on the antioxidant activity of sea buckthorn leaf tea. Therefore, ellagic acid and isorhamnetin‐3‐O‐neohesperidin can be used as potential quality markers for sea buckthorn leaf tea. This work provides a reference for taste improvement and quality control of sea buckthorn leaf tea.

a result, the Chinese government approved the use of sea buckthorn leaves as a common food ingredient in 2013. The development and application of sea buckthorn leaf as a raw material in food, medicine, animal feed, and even cosmetics have rapidly developed (Beveridge et al., 1999;Li, Liu, et al., 2021).
In recent years, scholars have focused on utilizing sea buckthorn leaves and converting them into a potential functional beverage product due to their health benefits. After drying at high temperatures, sea buckthorn leaf tea retains considerable nutritional value and is comparable with commonly consumed vegetables (Tanwar et al., 2018). The common forms of sea buckthorn leaf tea on the market are bulk sea buckthorn leaf tea (unpackaged) and bagged sea buckthorn leaf tea. Sea buckthorn leaf tea contains a variety of functional ingredients with many health benefits, such as lipid lowering, weight loss, and antioxidation (Cho et al., 2014;Lee et al., 2011).
Sea buckthorn leaf tea is an emerging commodity and is available in various forms such as loose sea buckthorn leaf tea and bagged sea buckthorn leaf tea when sold in the market. Since sea buckthorn is widely distributed in China, differences in ecological environment, genetic germplasm, and processing method may significantly affect the chemical composition and activity of sea buckthorn leaf tea (Tanwar et al., 2018). However, no suitable quality evaluation methods and standards are available yet for this product. As a potential functional beverage, the taste of sea buckthorn leaf tea has a direct impact on its market consumption and acceptance. Few reports are available on factors affecting the taste of sea buckthorn leaf tea. Therefore, sea buckthorn leaf tea currently available in the Chinese market should be investigated to establish quality evaluation methods and explore key factors affecting taste. In particular, the correlation between the phenolic composition of sea buckthorn leaves and their taste should be established to expand the utilization of sea buckthorn leaf tea as a potential health resource.
The aim of this study was to investigate the phenolic composition, sensory quality, and antioxidant activity of sea buckthorn leaf tea available in the Chinese market, to investigate the important factors affecting the taste of sea buckthorn leaf tea, and to provide valuable data for the quality evaluation of sea buckthorn leaf tea.

| Chemicals and materials
The following standards with purity higher than 98% were purchased from Yuanye Biotechnology Co., Ltd.: isoquercitrin,

| Sample materials
Sea buckthorn leaf tea samples (No. S1-S18) were purchased in the

| Preparation of extracts
In an ultrasonic extractor, about 1 g of tea powder was extracted with 25 ml of water at a temperature of 50°C and a power of 300 W for 40 min. The extracts were stored at 4°C prior to further assay.

| The content of flavonoids and tannins
In order to more fully reflect the content of polyphenols in sea buckthorn leaf tea, a semiquantitative method was temporarily adopted.
First, the chromatographic peaks of sea buckthorn leaf tea were identified based on the UV characteristic spectra of flavonoids and tannins (the UV absorption spectra of flavonoids in methanol mainly appear between 300 and 400 nm and 240 and 280 nm; and the tannin has strong characteristic absorption at about 275 nm).
Furthermore, the contents of individual flavonoids and tannins were calculated by comparing their peak areas with those of isoquercitrin and ellagic acid standards, respectively. Finally, the contents of all single flavonoid or tannin were added to obtain the total contents of flavonoids and tannins in sea buckthorn leaf tea.

| Antioxidant activity assays
In vitro antioxidant activity of ultrasonic extracts of sea buckthorn leaf tea was determined by FRAP, DPPH, and ABTS methods, respectively.

| FRAP method
Total antioxidant capacity was evaluated using the FRAP method described by . About 15 μl of sea buckthorn leaf tea solution was placed separately in a 96-well plate, mixed with 180 μl of FRAP working solution, and shaken thoroughly for 10 s. The solution was incubated for 6 min at 37°C, and absorbance was recorded at 734 nm. The results were expressed as mg TE/g. 2.6.2 | DPPH method DPPH method was used to determine the antioxidant activity with reference to literature (Gülçin et al., 2011;Köksal & Gülçin, 2008 was expressed as milligrams of Trolox equivalents per gram of dried sample (mg TE/g).

| ABTS method
The ABTS radical scavenging activity of sea buckthorn leaf tea was referenced from the method of . ABTS was bluegreen in color and had a characteristic absorbance at 734 nm. About 10 μl of sea buckthorn leaf tea solution was placed separately in a 96-well plate, mixed with 200 μl of ABTS + working solution, and shaken thoroughly for 10 s. The sample was incubated at room temperature for 5 min, and absorbance was recorded at 734 nm. The results were expressed as mg TE/g.

| Analysis of the antioxidant potency composite (APC) index
The APC index was used to evaluate the overall antioxidant activity of sea buckthorn leaf tea (Peng et al., 2019

| Sensory evaluation
Each sea buckthorn leaf tea (1:50, w/v) was steeped at 80°C for 5 min, and 5 ml of each tea infusion was poured into a disposable plastic cup and cooled to room temperature. Fifteen trained panelists including 11 women and 4 men aged between 20 and 45 years evaluated the product in the laboratory. Each person evaluated 18 sea buckthorn leaf tea samples. The following attributes were evaluated: color, aroma, sour, bitter, sweet, umami, astringent, and overall acceptance. The scoring criteria were based on the 10-component table method described in a previous work (Liu et al., 2018), as shown in Table S2. Each panelist was asked to wash their taste buds with drinking water between different samples at intervals of 1-2 min. Sensory profiles of the samples were developed based on their average score. Overall acceptance was evaluated based on the taste and color of sea buckthorn leaf tea. The standard references for bitterness, astringency, freshness, and sweetness were quinine hydrochloride, tannin, sodium glutamate, and sucrose, respectively.

| Statistical analysis
Standard deviation was calculated for each experiment after three repetitions. One-way analysis of variance (ANOVA, p < .05) with Tukey's HSD and Tamhane's test was used to evaluate the results using SPSS 20.0. Pearson's correlation coefficients were determined using SPSS 20.0.

| Composition and contents of phenolic compounds
An analytical UPLC-DAD method was developed for the simultaneous determination of 10 phenolic compounds in sea buckthorn leaf tea. The method was validated by determining linearity, precision, repeatability, stability, and recovery rates. Good linear correlations were obtained for the phenolic compounds using this method with R 2 > .999. Moreover, the relative standard deviations of the repeatability, precision, stability, and recovery of the method were all below 5.00%, and the recovery was within the range of 97.91%-102.70%.
The results confirm the validity of the method for the evaluation of sea buckthorn leaf tea (Table S1). The UPLC-DAD chromatogram of a representative sample S3 mixed with a standard solution is shown in Figure S1.
In this study, on the other hand, quercetin was higher in sea buckthorn leaf tea, which could be due to different processing methods and origins. Samples with higher isorhamnetin derivatives (isorhamnetin and isorhamnete-3-O-neohesperidin) were S15 (Chaoyang, Liaoning) and S11 (Urumqi, Xinjiang). The study reported that the isorhamnetin derivatives content of sea buckthorn leaves was superior to that of quercetin derivatives (Pop et al., 2013).
Among all the test samples, ellagic acid total content was the highest at 59.12 mg/g, consistent with the report that tannin components are mainly found in sea buckthorn leaves .
The study evaluated the sea buckthorn leaf profiles and reported that phenolic acid content is proportional to the flavonoid content (Raudone et al., 2021). However, in the present work, the samples with a more excellent ellagic acid content of sea buckthorn leaf tea all had lower total flavonoid content. This finding could be due to high-temperature enzyme inactivation, thus better preserving the phenolic acids in the sea buckthorn leaf tea (Ma et al., 2019). The contents of catechins and apigenin were low in the sea buckthorn leaf tea.
The content of sea buckthorn leaf tea polyphenolic compounds is shown in Figure 1. The content of tannins ranged from 12.93 to 2.28 mg/g and the content of flavonoids ranged from 12.02 to 4.26 mg/g. S1 (Xining, Qinghai, 11.98 mg/g), S2 (Dingxi Gansu, 10.09 mg/g), and S9 (Tacheng, Xinjiang, 10.53 mg/g) had higher flavonoid content. S1 (Xining, Qinghai, 12.93 mg/g), S2 (Dingxi, Gansu, 9.05 mg/g), and S6 (Da Hinggan Ling Prefecture, Heilongjiang, 7.12 mg/g) had higher tannin content. In addition, the flavonoid and tannin contents of S17 (Tongliao, Inner Mongolia) were low. S17 was identified as sea buckthorn leaf black tea. In general, black tea decreases flavonoids and flavonoid glycosides after a specific processing step during fermentation (Feng et al., 2020). The bagged sea buckthorn leaf tea (S1-S7, 7.35 ± 2.85 mg/g) contained higher levels of tannins than the bulk sea buckthorn leaf tea (S8-S18, 3.01 ± 0.59 mg/g; p < .01). However, there was no significant difference in total flavonoids content between the two types of tea (p > .05). This finding illustrates the higher degree of crushing in bagged tea than in bulk tea.
It also illustrates that proper crushing of tea leaves can increase the extraction rate of active ingredients (Danna et al., 2016).

| Analysis of antioxidant activity
According to literature reports, evaluation of the antioxidant activities of natural antioxidants is difficult using a single method.
The average APC index of the bagged sea buckthorn leaf tea (S1-S7, 80.59 ± 13.48) was found to be stronger than that of the bulk sea buckthorn leaf tea (S8-S18, 67.56 ± 11.60; p < .05). This result highlighted that the antioxidant capacity of the bagged sea buckthorn leaf tea was stronger than that of the loose sea buckthorn leaf tea (p < .05). We speculate that this might be due to the higher degree of grinding tea leaves for bagged tea than that for loose sea buckthorn leaf tea; the degree of grind is positively correlated with antioxidant capacity (Q. Xu et al., 2021).

| Sensory evaluation
Taste is one of the important quality factors that influence consumer preferences after variety . Therefore, sensory analysis of sea buckthorn leaf tea was carried out. The experiment is illustrated in Figure 2. Color is an important quality criterion that influences consumer preferences because it is the first attribute perceived by consumers (Lima et al., 2019). The results showed that sea buckthorn leaf tea infusion was yellow-brown, and the color difference was not significant (p > .05). The common explanation for the yellowish-brown color of tea infusion is that it is determined F I G U R E 1 Polyphenols content of sea buckthorn leaf tea by water-soluble flavonoids such as kaempferol, isoquercitrin, and rutin. The result showed no significant difference in the aroma evaluation of the sea buckthorn leaf teas.
Flavor is one of the important factors of acceptance among consumers. Astringency was found in all tea infusion from 1.29 ± 1.39 to 5.64 ± 1.62. The sweet scores pattern showed the highest score at S18 (Tongliao, Inner Mongolia, 3.38), followed by S4 (Xinzhou, Shanxi, 1.24). Acid flavor was found in all tea infusion from 1.09 ± 1.63 to 3.04 ± 2.36. The fresh scores pattern showed the highest score at S18 (Tongliao, Inner Mongolia, 3.26), followed by S4 (Xinzhou, Shanxi, 1.24). Bitterness was found in all tea infusion from 2.00 ± 1.53 to 5.96 ± 2.23. Based on the score of the sensory characteristics, the corresponding radar chart was created. Statistical differences were found in bitterness, astringency, and acid in sea buckthorn leaf tea samples (p < .05). Ma et al. (2019) claimed that the astringency perception threshold of sea buckthorn leaves is low. However, their results were inferred from this fact. This fact is that sea buckthorn leaves are rich in flavonol glycosides, which have little effect on astringency (Scharbert & Hofmann, 2005). Therefore, our experimental results on the Note: Values are expressed as the mean standard deviation, n = 3. Significant differences between different drying methods of each variety (p < .05) are marked with a-h. TA B L E 3 Antioxidant activity of sea buckthorn leaf tea F I G U R E 2 Sea buckthorn leaf tea taste evaluation chart predominantly bitter and astringent taste of sea buckthorn leaf tea are more reliable. It is currently reported that the astringent substances of tea are mainly polyphenols, and the primary phenolic acids in tea are ellagic acid, chlorogenic acid, etc. (Ceci et al., 2018).
At the same time, flavonoids are one of the important factors affecting the bitter taste of tea . At present, technologies to improve the bitter taste of tea mainly include enzyme treatment, microcapsule technology, food irradiation, and other technologies (Ye et al., 2022). Therefore, these methods can be used to improve the taste of sea buckthorn leaf tea.
Acceptability was found in all 18 tea infusion from 2.27 ± 1.62 to 6.24 ± 1.56. The top three teas with the acceptability were S11 (Urumqi, Xinjiang, 6.36), S17 (Tongliao, Inner Mongolia, 6.03), and S5 (Lvliang, Shanxi, 5.67). S8 (Xinjiang, 2.15) has the lowest acceptance. According to Figure 2, it can be shown that the bitterness and astringency of sea buckthorn leaf tea are closely related to the overall acceptability. Meanwhile, the mean value of acceptability of the bagged sea buckthorn leaf tea (S1-S7) was 5.8 and the mean value of acceptability of the bulk sea buckthorn leaf tea (S8-S10) was 5.4.
Hence, bagged tea is more popular among consumers. In summary, the results showed the influence of taste and packaging form on the acceptance of sea buckthorn leaf tea.

| Correlation analysis
To explore the relationship between antioxidant activity, sensory evaluation, and individual phenols and flavonoids, we calculated the Pearson's correlation coefficient (Tables 4 and 5). This is because polyphenolic compounds are an important factor affecting the antioxidant properties and taste of sea buckthorn leaf tea (Barbe et al., 2019;Delius et al., 2017). Note: Pearson's correlation at **p < .01, *p < .05. The larger the correlation coefficient is, the redder the cell color is; the smaller the correlation coefficient, the greener the color.

TA B L E 4 Correlations between antioxidant activity and main compounds
As shown in As shown in Table 5

| CON CLUS ION
In this study, sea buckthorn leaf teas circulating in the China market were analyzed by UPLC, sensory evaluation, and in vitro antioxidant assay.
The results indicate that the quality of sea buckthorn leaf tea varies greatly, and the polyphenol content, antioxidant activity, and sensory evaluation of bagged sea buckthorn leaf tea were better than those of bulk sea buckthorn leaf tea. Importantly, the taste astringency of sea buckthorn leaf tea was closely related to ellagic acid and isorhamnetin-3-O-neohesperidin. Meanwhile, isorhamnetin-3-O-neohesperidin had a greater effect on the antioxidant activity of sea buckthorn leaf tea.
Therefore, ellagic acid and isorhamnetin-3-O-neohesperidin are recommended as quality control indicators for sea buckthorn leaf tea. The study provides a reference for the taste improvement of sea buckthorn leaf tea and valuable data for the quality control of sea buckthorn leaf tea. It is beneficial for promoting and applying sea buckthorn leaf tea as potential functional beverage products.

ACK N OWLED G M ENTS
The authors thank the support from the Academician and Experts Workstation of Sea-buckthorn Industry in LAN County, Shanxi Province.

CO N FLI C T O F I NTE R E S T
All authors disclosed no relevant relationships.

DATA AVA I L A B I L I T Y S TAT E M E N T
Even though adequate data have been given in the form of tables and figures, all authors declare that if more data are required, then the data will be provided on request basis.

E TH I C S S TATEM ENT
This article does not contain any studies performed with human participants or animals by any of the authors.

CO N S E NT TO PA RTI CI PATE
Corresponding and all the coauthors are willing to participate in this manuscript.

CO N S E NT FO R PU B LI C ATI O N
All authors are willing for publication of this manuscript.